UMR3244 – Dynamique de l’information génétique

Publications de l’équipe

Année de publication : 2005

Maria A Cerone, Chantal Autexier, J Arturo Londoño-Vallejo, Silvia Bacchetti (2005 Aug 24)

A human cell line that maintains telomeres in the absence of telomerase and of key markers of ALT.

Oncogene : 7893-901 En savoir plus
Résumé

In human somatic cells proliferation results in telomere shortening due to the end replication problem and the absence of adequate levels of telomerase activity. The progressive loss of telomeric DNA has been associated with replicative senescence. Maintenance of telomere structure and function is, therefore, an essential requisite for cells that proliferate indefinitely. Human cells that have acquired the immortal phenotype mostly rely on telomerase to compensate for telomere shortening with cell division. However, a certain percentage of immortalized cell lines and human tumors maintain their telomeres by Alternative Lengthening of Telomeres (ALT), a mechanism not fully understood but apparently based on homologous recombination. Here, we report the isolation of an immortal human cell line that is derived from an ALT cell line but maintains telomeres in the absence of key features of ALT and of telomerase. The properties of these cells suggest that the identification of ALT cells may not be reliably based on known ALT markers. This finding is of relevance for discriminating between the mortal and immortal phenotype among telomerase-negative cells in vitro and in vivo, particularly in regard to the development of pharmacological approaches for cancer treatment based on telomerase inhibition.

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Maria Antonietta Cerone, Ryan J Ward, J Arturo Londoño-Vallejo, Chantal Autexier (2005 Mar 9)

Telomerase RNA mutated in autosomal dyskeratosis congenita reconstitutes a weakly active telomerase enzyme defective in telomere elongation.

Cell cycle (Georgetown, Tex.) : 585-9 En savoir plus
Résumé

Dyskeratosis congenita (DC) is a rare multi-system syndrome characterized by nail dystrophy, abnormal skin pigmentation and mucosal leukoplakia. The gene mutated in the X-linked form of human DC encodes for dyskerin, a nucleolar pseudourydilase that is involved in rRNA maturation. Dyskerin is also involved in telomerase function through its interaction with the telomerase RNA (hTR). Mutations in dyskerin result in low levels of hTR, decreased telomerase activity and telomere shortening. Autosomal dominant DC is characterized by mutations in hTR, supporting the hypothesis that the DC phenotype may be caused by impaired telomere maintenance. Several mutations have been identified in different regions of hTR in patients affected by autosomal dominant DC. Recent reports have shown that coexpression of wild-type hTR with hTR harboring mutations found in the pseudoknot domain does not affect telomerase activity in vitro. However, these studies did not assess the consequences of mutant hTR expression at the telomeres. Here we provide the first direct in vivo evidence that a mutant hTR carrying the GC to AG double substitution in the pseudoknot at nucleotides 107-108 found in patients affected by autosomal dominant DC does not behave as a dominant-negative for telomere maintenance. Rather it reconstitutes a weakly active telomerase enzyme, which is defective in telomere elongation.

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Silvia Prieler, Alexandra Penkner, Valérie Borde, Franz Klein (2005 Jan 19)

The control of Spo11’s interaction with meiotic recombination hotspots.

Genes & development : 255-69 En savoir plus
Résumé

Programmed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11. Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae. Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells. The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80. In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11’s interaction to the core region of the hotspot. In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene. In both rad50S and com1Delta/sae2Delta mutants, we observed a novel reaction intermediate between Spo11 and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking. Although this DNA does not contain a break, its recovery requires Spo11’s catalytic residue Y135. We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1Delta/sae2Delta mutants transiently arrest.

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Année de publication : 2004

Valérie Borde, Waka Lin, Eugene Novikov, John H Petrini, Michael Lichten, Alain Nicolas (2004 Feb 18)

Association of Mre11p with double-strand break sites during yeast meiosis.

Molecular cell : 389-401 En savoir plus
Résumé

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.

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Année de publication : 2003

Hajime Murakami, Valerie Borde, Takehiko Shibata, Michael Lichten, Kunihiro Ohta (2003 Jul 11)

Correlation between premeiotic DNA replication and chromatin transition at yeast recombination initiation sites.

Nucleic acids research : 4085-90 En savoir plus
Résumé

The DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae are preceded first by DNA replication and then by a chromatin transition at DSB sites. This chromatin transition, detected as a quantitative increase in micrococcal nuclease (MNase) sensitivity, occurs specifically at DSB sites and not at other MNase-sensitive sites. Replication and DSB formation are directly linked: breaks do not form if replication is blocked, and delaying replication of a region also delays DSB formation in that region. We report here experiments that examine the relationship between replication, the DSB-specific chromatin transition and DSB formation. Deleting replication origins (and thus delaying replication) on the left arm of one of the two parental chromosomes III affects DSBs specifically on that replication-delayed arm and not those on the normally replicating arm. Thus, replication timing determines DSB timing in cis. Delaying replication on the left arm of chromosome III also delays the chromatin transition at DSB sites on that arm but not on the normally replicating right arm. Since the chromatin transition precedes DSB formation and requires the function of many genes necessary for DSB formation, these results suggest that initial events for DSB formation in chromatin are coupled with premeiotic DNA replication.

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Année de publication : 2000

V Borde, A S Goldman, M Lichten (2000 Oct 29)

Direct coupling between meiotic DNA replication and recombination initiation.

Science (New York, N.Y.) : 806-9 En savoir plus
Résumé

During meiosis in Saccharomyces cerevisiae, DNA replication occurs 1. 5 to 2 hours before recombination initiates by DNA double-strand break formation. We show that replication and recombination initiation are directly linked. Blocking meiotic replication prevented double-strand break formation in a replication-checkpoint-independent manner, and delaying replication of a chromosome segment specifically delayed break formation in that segment. Consequently, the time between replication and break formation was held constant in all regions. We suggest that double-strand break formation occurs as part of a process initiated by DNA replication, which thus determines when meiotic recombination initiates on a regional rather than a cell-wide basis.

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Année de publication : 1999

V Borde, T C Wu, M Lichten (1999 Jun 22)

Use of a recombination reporter insert to define meiotic recombination domains on chromosome III of Saccharomyces cerevisiae.

Molecular and cellular biology : 4832-42 En savoir plus
Résumé

In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two « hot » regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the « cold » properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis.

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Année de publication : 1998

V Borde, M Duguet (1998 Jun 20)

DNA topoisomerase II sites in the histone H4 gene during the highly synchronous cell cycle of Physarum polycephalum.

Nucleic acids research : 2042-49 En savoir plus
Résumé

The nearly perfect synchrony of nuclear division in a plasmodium of Physarum polycephalum provides a powerful system to analyze topoisomerase II cleavage sites in the course of the cell cycle. The histone H4 locus, whose schedule of replication and transcription is precisely known, was chosen for this analysis. Drug-induced topoisomerase II sites are clustered downstream of the histone H4 gene and appear highly dependent on cell cycle stage. They were only detected in mitosis and at the very beginning of S phase, precisely at the time of replication of the histone H4 region. The sites, which were absent in G2 phase, reappeared at the next mitosis. Remarkably, DNase I hypersensitive sites occurred in nearly the same location, but their schedule was totally different: they were absent in mitosis and present in G2. This schedule follows H4 transcription, which peaks in mid-S phase and in the second part of G2 phase and is off during mitosis. These results suggest that topoisomerase II may not be involved in transcription, but plays a role in remodeling chromatin structure, both during chromosome condensation in prophase/metaphase to allow their decatenation and during chromosome decondensation after metaphase to allow replication fork passage throughout the region.

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